Efforts to produce cells in vitro have met with limited success. While embryonic stem cells can be expanded indefinitely, it is difficult to expand differentiated cells. Moreover, it is currently not cost-effective to produce differentiated cells from stem cells in vitro.
The growth of foreign cells within an animal would provide substantial value in biotechnology. The production, expansion and isolation of cells using a non-human mammalian host would provide cells for infusion and transplantation, the production of drugs and factors for therapy, cells for tissue engineering and assays. The production of animals that are chimeric, and contain foreign cells would be useful for transplantation, models of disease, and for the functional assessment of a transgene.
Two factors make it challenging to grow foreign cells in animals, however. First, the foreign cells normally would be rejected by the host animal. Second, the foreign cells would need to compete with the native cells of the animal.
Cells have been grown in congenitally immune deficient animals. For example, human lymphocytes have been grown within SCID (severe combined immune deficiency) mice (1). These mice normally have a deficiency of B and T cells. However, the human lymphocytes are not appropriately functional and do not provide a normal immune response (2, 3).
Transgenic mice have been used to enhance engraftment with foreign cells. Rhim and Brinster produced transgenic mice with a defective urokinase plasminogen activator gene controlled by an albumin promoter. The native hepatocytes in these mice were defective and did not survive long. The defective hepatocytes were eventually replaced when foreign hepatocytes, including rat hepatocytes were injected (4). This model is not practical, however. Mice cannot be used as a source of donor organs. In addition, the pups had hypofibrinogenemia and usually died of neonatal hemorrhage (5).
SCID mice that are homozygous for urokinase plasminogen activator (uPA) have been engrafted with human hepatocytes (6). Due to the death of the mouse hepatocytes, the homozygous mice are difficult to keep alive. Heterozygous mice must be bred, and the homozygous offspring transplanted right after birth. The mice often die of liver failure before the human hepatocytes provide support. Because they lack a functional immune system, however this model has limited value for the development of vaccines.
Braun et al. used adult transgenic mice containing the suicide gene thymidine kinase to enhance engraftment with foreign cells (7). The thymidine kinase was under control of an albumin promoter and was expressed in the hepatocytes. The hepatocytes were normal until the prodrug gancyclovir was administered to adult mice. Most of the hepatocytes then died off, leading to regeneration with new hepatocytes. This system was an improvement over the uPA mouse model, because it permits controlled killing and turnover of the hepatocytes. But while engraftment was enhanced, overall survival was not generally improved. Following hepatic necrosis, most mice did not survive long enough to allow the differentiation and organization of the new hepatocytes. The small size of the mouse also limits the application of this system, because chimeric livers could not be produced for human transplantation.
Foreign cells have been infused into fetal animals, leading to limited engraftment. For example, limited engraftment of hematopoietic cells has been demonstrated in fetal sheep and monkeys (8) and by infusion into fetal mice, sheep, and pigs (9, 10, 11). Infusion of human hepatocytes or stem cells into fetal pigs has resulted in only limited engraftment (12). The intrauterine environment is favorable to engraftment with foreign cells, and the host naturally develops immune tolerance to the cells (13). The uterine environment is also naturally sterile. However, engraftment of foreign cells is very limited due to competition with the native host cells.
While transgenic mice can be readily produced to study diseases related to a specific gene, it is not practical to produce large animals. For example, pigs are several thousand times larger than mice and their generational time is about 10 times as long. To date, there has only been one herd of transgenic pigs produced for the study of human disease, retinitis pigmentosa (Petters et al., Nature Biotechnology 15, 965, 1997).
Thus, there is a need in the art for methods of engrafting foreign cells in fetal host animals.